Process for producing 4-thiouracil

ABSTRACT

A novel microbiological process for preparing 4-thiouracil, a known and useful compound. The compound is produced by the controlled fermentation of the new microorganism Streptomyces libani subsp. soldani, NRRL 8173, and its strains W and P having the NRRL designations NRRL 8174 and NRRL 8175, respectively.

BACKGROUND OF THE INVENTION

The compound, 4-thiouracil, produced by the novel process of the subject invention has been known since about 1945. It has been described in the literature as an antibacterial, antitumor agent, useful in photographic processes and as a plant growth stimulator. The compound has been prepared in the prior art by chemical means. There is no known prior art which describes the production of 4-thiouracil by a microbiological process.

BRIEF SUMMARY OF THE INVENTION

The novel process of the subject invention produces a high yield of the compound 4-thiouracil. This process is conducted by culturing the new microorganism Streptomyces libani subsp. soldani, NRRL 8173, and its strains NRRL 8174 and NRRL 8175, in an aqueous nutrient medium under aerobic conditions. 4-Thiouracil is isolated from the fermentation beer by procedures generally known in the antibiotic art, for example, filtration, solvent extraction, partition chromatography, adsorption onto charcoal and the like.

DETAILED DESCRIPTION OF THE INVENTION

The compound produced by the novel process of the subject invention, 4-thiouracil, can be shown by the following structural formula: ##STR1##

This compound is active against a variety of bacteria, for example, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Proteus vulgaris. Accordingly, 4-thiouracil can be used in various environments to eradicate or control these microorganisms. For example, it can be used for treating breeding places of silkworms and to prevent or minimize infections caused by Bacillus subtilis. Since 4-thiourcil is active against E. coli, it can be used to reduce, arrest, and eradicate slime production in papermill systems caused by this microorganism.

THE MICROORGANISM

The microorganism used for the production of 4-thiouracil is Streptomyces libani subsp. soldani. A subculture of this microorganism can be obtained from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A. Its accession number in this depository is NRRL 8173. It should be understood that the availability of the culture does not constitute a license to practice the subject invention in derogation of patent rights granted with the subject instrument by governmental action.

The microorganism of this invention was studied and characterized by Alma Dietz of the Upjohn Research Laboratories.

A new soil isolate NRRL 8173 has been compared with two strains and with the type culture Streptomyces libani. The new culture was considered to be most similar to S. libani in its color pattern on Ektachrome and in its microscopic characteristics. Detailed comparisons are given in Tables 1-5. A number of isolates of S. libani are characterized by Baldacci and Grein [Baldacci, E. and A. Grein. 1966. Streptomyces avellaneus and Streptomyces libani; two new species characterized by a hazel-nut brown (avellaneus) aerial mycelium. Giornale di Microbiologia 14: 185-198]. The characterizations reveal the natural variability of the culture. The same variability is noted between NRRL 8173 and the strains NRRL 8174 and NRRL 8175. S. libani var. soldani may be placed in the Gray series of Table 17.42f (Part 17. Actinomycetes and Related Organisms) of the Eighth Edition of Bergey's Manual of Determinative Bacteriology [Buchanan, R. E., and N. E. Gibbons. 1974. Bergey's Manual of Determinative Bacteriology, Eight Edition, The Williams and Wilkins Co., Baltimore]. The type culture, S. libani, is listed in Table 17.42f. An additional characterization of S. libani is found in Shirling and Gottlieb [Shirling, E. B. and D. Gottlieb. 1972. Cooperative description of type strains of Streptomyces. V. Additional descriptions. Int. J. Syst. Bacteriol. 22: 265-394]. Strong similarities are noted between the new soil isolate and S. libani in Tables 1-5 and in the references cited. The natural variability of the cultures and, in particular, the difference in antibiotic production require the new isolate to be considered a new subspecies. Therefore, in accordance with the rules set forth in the International Code of Nomenclature of Bacteria [International Code of Nomenclature of Bacteria, 1976 Revision. 1975. Edited by S. P. LaPage, p. H. A. Sneath, E. F. Lessel, V. B. D. Skerman, H. P. R. Seeliger and W. A. Clark. Published for the International Association of Microbiological Societies by the American Society for Microbiology, Washington, D.C.]the name Streptomyces libani subsp. soldani Dietz subsp. nova is proposed.

Color Characteristics

The cultures are melanin-negative and have white and gray aerial growth. Color characteristics of the cultures on Ektachrome are given in Table 1. Reference color characteristics are given in Table 2. The cultures may be placed in the White (W) or Gray (GY) color groups of Tresner and Backus [Tresner, H. D. and E. J. Backus. 1963. System of color wheels for streptomycete taxonomy. Appl. Microbiol. 11:335-338].

Microscopic Characteristics

Spores with a smooth surface are found in spiral spore chains. More than 10 spores occur per chain. The spore shape may be oval, reniform or spherical. Microscopic determinations were made following the procedures of Pridham et al. [Pridham, T. G., C. W. Hesseltine and R. G. Benedict. 1958. A guide for the classification of streptomycetes according to selected groups. Placement of strains in morphological sections. Appl. Microbiol. 6:52-79] and Dietz and Mathews [Dietz, A. and J. Mathews. 1971. Classification of Streptomyces spore surfaces into five groups. Appl. Microbiol. 21:527-533].

Cultural and Biochemical Characteristics

Cultural and biochemical characteristics are cited in Table 3.

Carbon Utilization

Carbon utilization is cited in Tables 4 and 5.

Temperature

The cultures grew well in the temperature range of 18° -37° C. Growth did not occur at 45° or 55° C. Media used for temperature studies were Bennett's, Czapek's sucrose, maltose-tryptone and Hickey-Tresner agars.

Antibiotic-Producing Properties

S. libani subsp. soldani NRRL 8173produces 4-thiouracil. S. libani UC-5629 produces the libanomycin complex.

Source: Soil.

Type Culture: Streptomyces libani Baldacci and Grein, UC-5629, IMRU 3915.

Variety: Streptomyces libani subsp. soldani Dietz subsp. nova.

Strains: Streptomyces libani subsp. soldani strain W Streptomyces libani subsp. soldani strain P

                                      Table 1                                      __________________________________________________________________________     Appearance of Streptomyces libani subsp. soldani NRRL 8173 and the             Strains NRRL 8174 and NRRL 8175 vs. S. libani UC-5629 on Ektachrome*                   Determi-                                                               Agar Medium                                                                            nation                                                                               NRRL 8173                                                                               NRRL 8174                                                                              NRRL 8175                                                                               UC-5629                                __________________________________________________________________________     Bennett's                                                                              S    White     Lavender-gray                                                                          Trace gray                                                                             Lavender-gray                                   R    Pale yellow cream                                                                        Yellow  Pale tan                                                                               Pale yellow-tan                         Czapek's                                                                               S    White     White   Fair white                                                                             White                                   sucrose R    Cream     Pale yellow                                                                            Pale tan                                                                               Pale yellow                             Maltose S    White     Gray white                                                                             Fair white                                                                             Lavender-gray                           tryptone                                                                               R    Pale yellow                                                                              Yellow-tan                                                                             Pale yellow-                                                                           Pale yellow-tan                                                        tan                                             Peptone-iron                                                                           S    Trace white                                                                              Trace white                                                                            --      Trace lavender-                                                                gray                                            R    Yellow-tan                                                                               Yellow  Yellow-tan                                                                             Yellow                                  0.1% Tyrosine                                                                          S    Trace white                                                                              Trace white                                                                            --      Trace lavender-                                                                gray                                            R    Red-pink  Red-pink                                                                               Deep red                                                                               Pale yellow                             Casein strarch                                                                         S    White     Pale gray                                                                              Pale cream                                                                             Trace lavender-                                                                gray                                            R    Very pale cream                                                                          Pale yellow                                                                            Lavender gray                                                                          Pale yellow                             __________________________________________________________________________      *Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete             classification. Ann. N. Y. Acad. Sci. 60:152-154.   S = Surface R =            NOTE: "UC" is a registered trademark and refers to The Upjohn Company          Culture Collection.                                                      

                                      Table 2                                      __________________________________________________________________________     Reference Color Characteristics of S. libani subsp. soldani vs. S.             libani                                                                                 De-                                                                            ter-                                                                           mi-                                                                               ISCC-NBS Color-Name Charts Illustrated with Centroid Colors*                na-                                                                               S. libani var. soldani                                              Agar Medium                                                                            tion                                                                              NRRL 8173              NRRL 8174                                    __________________________________________________________________________     Bennett's                                                                              S  86  l.Y  Light Yellow  93  y Gray                                                                              Yellowish gray                                     under                                                                      214 p.V  Pale violet                                                        R  84  s.Y  Strong yellow 89  p.Y  Pale yellow                                 P  84  s.Y  Strong yellow 90  gy. Y                                                                               Grayish yellow                      Czapek's                                                                               S  9   pk White                                                                            Pinkish white 263 White                                                                               White                               sucrose R  52  l.0  Light orange  73  p.OY Pale orange yellow                          P  28  l.y Pink                                                                            Light yellowish pink                                                                         70  l.OY Light orange yellow                 Maltose-                                                                               S  93  y Gray                                                                              Yellowish gray                                                                               263 White                                                                               White                               tryptone                                                                               R  83  brill.Y                                                                             Brilliant yellow                                                                             89  p.Y  Pale yellow                                 P  --                     71  m OY Moderate orange yellow              Hickey- S  92  y White                                                                             Yellowish white                                                                              9   pk White                                                                            Pink white                          Tresner R  87  m.Y  Moderate yellow                                                                              70  l.OY Light orange yellow                         P  --                     71  m OY Moderate orange yellow              Yeast extract-                                                                         S  86  l.Y  Light yellow  263 White                                                                               White                               malt extract   under                                                           (ISP-2)    294 p.V  Pale violet                                                        R  91  d.gy.Y                                                                              Dark grayish yellow                                                                          70  l.OY Light orange yellow                         P  --                     --                                           Oatmeal S  261 l.gy.p R                                                                            Light grayish purplish red                                                                   93  y Gray                                                                              Yellowish gray                      (ISP-3) R  89  p.Y  Pale yellow   89  p.Y  Pale yellow                                 P  --                     90  gy.Y Grayish yellow                      Inorganic                                                                              S  86  l.Y  Light yellow  93  y Gray                                                                              Yellowish gray                      salts-starch   over                                                            (ISP-4)    244 p.r P                                                                               Pale reddish purple                                                R  83  brill. Y                                                                            Brilliant yellow                                                                             89  p.Y  Pale yellow                                 P  87  m.Y  Moderate yellow                                                                              90  gy.Y Grayish yellow                      Glycerol-                                                                              S  89  p.Y  Pale yellow   265 med. Gy                                                                             Medium gray                         asparagine     over                                                            (ISP-5)    261 l.gy.p R                                                                            Light grayish purplish red                                         R  87  m.Y  Moderate yellow                                                                              89  p.Y  Pale yellow                                 P  87  m.Y  Moderate yellow                                                                              90  gy.Y Grayish yellow                              De-                                                                            ter-                                                                           mi-                                                                               ISCC-NBS Color-Name Charts Illustrated with Centroid Colors*                na-                                                                               S. libani var. soldani S. libani                                    Agar Medium                                                                            tion                                                                              NRRL 8175              UC-5629                                      __________________________________________________________________________     Bennett's                                                                              S  93  y Gray                                                                              Yellowish gray                                                                               93  y Gray                                                                              Yellowish gray                              R  70  l.OY Light orange yellow                                                                          67  brill.OY                                                                            Brilliant orange yellow                     P  67  brill.OY                                                                            Brilliant orange-yellow                                                                      68  s.OY Strong orange yellow                Czapek's                                                                               S  92  y White                                                                             Yellowish white                                                                              253 White                                                                               White                               sucrose R  76  l.y Br                                                                              Light yellowish brown                                                                        73  p.OY Pale orange yellow                          P  77  l.y Br                                                                              Light yellowish brown                                                                        29  m.y Pink                                                                            Moderate yellowish pink             Maltose-                                                                               S  92  y White                                                                             Yellowish white                                                                              93  y Gray                                                                              Yellowish gray                      tryptone                                                                               R  89  p.Y  Pale yellow   83  brill. Y                                                                            Brilliant yellow                            P  76  l.y Br                                                                              Light yellowish brown                                                                        84  s.Y  Strong yellow                       Hickey- S  92  y White                                                                             Yellowish white                                                                              9   pk White                                                                            Pinkish white                       Tresner R  73  p.OY Pale orange yellow                                                                           73  p.O  Pale orange yellow                          P  76  l.y Br                                                                              Light yellowish brown                                                                        67  brill.OY                                                                            Brilliant orange yellow             Yeast extract-                                                                         S  9   pk White                                                                            Pinkish white 263 White                                                                               White                               malt extract                                                                   (ISP-2)                                                                                R  57  l.Br Light brown   70  l.OY Light orange yellow                         P  42  l.r Br                                                                              Light reddish brown                                                                          71  m.OY Moderate orange yellow              Oatmeal S  93  y Gray                                                                              Yellowish gray                                                                               93  y Gray                                                                              Yellowish gray                      (ISP-3) R  70  l.OY Light orange yellow                                                                          89  p.Y  Pale yellow                                 P  71  m.OY Moderate orange yellow                                                                       101 1.Gray                                                                              light gray                          Inorganic                                                                              S  92  y White                                                                             Yellowish white                                                                              81  d.gy.yBr                                                                            Dark grayish yellowish              salts-starch                               brown                               (ISP-4)                                                                                R  70  l.OY Light orange yellow                                                                          73  p.OY Pale orange yellow                          P  71  m.OY Moderate orange yellow                                                                       73  d.OY Dark orange yellow                  Glycerol-                                                                              S  93  y Gray                                                                              Yellowish gray                                                                               9   pk White                                                                            Pinkish white                       asparagine                                                                     (ISP-5)                                                                                R  70  l.OY Light orange yellow                                                                          73  p.OY Pale orange yellow                          P  71  m.OY Moderate orange yellow                                                                       71  m.OY Moderate orange                     __________________________________________________________________________                                                yellow                               *Kelly, K. L. and D. B. Judd. 1955. The ISCC-NBS method of designating         colors and a dictionary of color names. U.S. Dept. Comm. Circ. 553.            S = Surface R = Reverse P = Pigment                                      

                                      Table 3                                      __________________________________________________________________________     Cultural and Biochemical Characteristics                                                 Determi-          S. libani subsp. soldani                                                                              S. libani                   Medium    nation                                                                               NRRL 8173      NRRL 8174                                                                              NRRL 8175   UC-5629                     __________________________________________________________________________     Agar                                                                           Peptone-iron                                                                             S     --          White      --          Trace pale gray                       R     Colorless   Yellow     Yellow-tan  Pale cream-tan                        P     --          --         --          --                                    O     Melanin negative                                                                           Melanin negative                                                                          Melanin negative                                                                           Melanin negative            Calcium malate                                                                           S     Fair white  Trace white                                                                               --          Trace pale gray                       R     White or colorless                                                                         Colorless  Colorless   Colorless or pale                                                              gray                                  P     --          --         --          --                                    O     Malate solubilized                                                                         Malate not solu-                                                                          Malate not solu-                                                                           Malate not solu-                                        bilized    bilized     bilized                     Glucose   S     Cream       White with trace                                                                          Trace white Pale gray-white             asparagine                  gray                                                         R     Pink-tan    Cream      Pale tan    Pale cream-pink                       P     Pink        --         Trace pink  Pale pink                   Skim milk S     Trace white on edge                                                                        White on edge                                                                             --          Pale gray-white                       R     Pink-orange Colorless  Colorless and                                                                              Orange-tan                                                         trace orange                                      P     Pink-orange --         --          Orange-tan                            O     Casein hydrolyzed                                                                          Casein not Casein not  Casein not                                              hydrolyzed hydrolyzed to                                                                              hydrolyzed                                                         trace hydrolyzed                                                               around growth                           Tyrosine  S     Gray        Pale cream-pink                                                                           --          Pale cream-white                      R     Red-tan     Light red-tan                                                                             Red-tan     Yellow                                P     Red-tan     Light red-tan                                                                             Red-tan     Pale yellow                           O     Tyrosine solu-                                                                             Tyrosine solu-                                                                            Tyrosine solu-                                                                             Tyrosine solu-                              bilized     bilized    bilized     bilized                     Xanthine  S     Cream white Pale cream white                                                                          Fair pale gray                                                                             Cream white                           R     Cream       Cream      Cream       Pale yellow                           P     Pale cream  Pale cream Cream       Pale yellow                           O     Xanthine not                                                                               Xanthine not                                                                              Xanthine solu-                                                                             Xanthine solu-                              solubilized solubilized                                                                               bilized     bilized                     Nutrient starch                                                                          S     Cream white Cream white                                                                               --          Cream white                           R     Cream       Cream      Cream       Pale yellow                           P     --          Pale Cream Pale cream  --                                    O     Starch solubilized                                                                         Starch solubilized                                                                        Starch solubilized                                                                         Starch solubilized          Yeast extract-                                                                           S     Cream       Pale gray-cream                                                                           --          Pale gray-cream             malt extract                                                                             R     Yellow-tan  Pale cream-brown                                                                          Pale yellow-tan                                                                            Yellow-tan                            P     --          Pale olive-gray                                                                           Pale yellow-tan                                                                            Yellow-tan                  Peptone-yeast                                                                            S     White       Cream white                                                                               --          Very pale gray-white        extract-iron                                                                             R     Yellow      Pale tan   Yellow-tan  Yellow-tan                  (ISP-6)   P     --          --         Pale yellow-tan                                                                            Yellow-tan                  L-Tyrosine                                                                               S     Lavender-gray-                                                                             Lavender-gray-                                                                            Pale lavender-                                                                             Cottony pale-gray           (ISP-7)         white       cream with gray-cream  with white flecks                                       black exudate                                                R     Pale tan    Pale olive Olive-tan   Cream-tan (reddish                                                             at base                               P     Pale tan    Pale olive Pale olive-tan                                                                             --                          Gelatin                                                                        Plain     S     Colorless surface                                                                          White surface                                                                             Colorless surface                                                                          Colorless surface                           ring        pellicle   ring        ring                                  P     --          Olive      --          Olive brown to olive                  O     Liquefaction                                                                               No liquefaction                                                                           No liquefaction                                                                            Liquefied in olive-                         complete                           brown area = trace                                                             liquefaction                Nutrient  S     Colorless surface                                                                          White surface                                                                             Colorless surface                                                                          Colorless surface                           ring        pellicle   ring        ring                                  P     --          Pale tan   --          --                                    O     Liquefaction                                                                               No liquefaction                                                                           No liquefaction                                                                            No liquefaction                             complete                                                       Nitrate Broth                                                                  Synthetic S     White aerial on                                                                            --         --          --                                          surface ring                                                             P     --          --         --          --                                    O     Trace flocculent                                                                           Trace flocculent                                                                          Flocculent bottom                                                                          Compact bottom                              bottom growth                                                                              bottom growth                                                                             growth      growth                      Synthetic O     Nitrate not Nitrate not                                                                               Nitrate not Nitrate not                                 reduced     reduced    reduced     reduced                                     Red with Zn dust                                                                           Red with zn dust                                                                          Red with zn dust                                                                           Red with zn dust            Nutrient  S     Trace white aerial                                                                         Pale gray-white                                                                           --          --                                          growth on   aerial growth                                                      surface ring                                                                               on surface ring                                              P     --          --         --          --                                    O     Flocculent bottom                                                                          Flocculent bottom                                                                         Pelliate like                                                                              Flocculent bottom                           growth      growth     bottom growth                                                                              growth                                      Nitrate not Nitrate not                                                                               Nitrate not Nitrate not                                 reduced     reduced    reduced     reduced                                     Orange with Zn                                                                             Orange with zn                                                                            Orange with zn                                                                             Orange with zn                              dust        dust       dust        dust                        Litmus milk                                                                              S     Pale gray-pink                                                                             Pale lavender-gray                                                                        --          Pale gray-pink                              aerial growth on                                                                           aerial growth on       aerial growth                               pink-maroon pale salmon            reddish-purple                              surface ring                                                                               surface ring           surface ring                          P     --          --         --          --                                    O     Peptonization 2/3                                                                          No peptonization                                                                          Peptonization 2/3                                                                          Peptonization 2/3                           Litmus reduced                                                                             Litmus changed to      Partial decoloriza-                         and coagulation                                                                            lavender gray          tion                                        1/3                                                                            pH 6.5      pH 7.1     pH 6.5      pH 6.7                      __________________________________________________________________________      S = Surface                                                                    R = Reverse                                                                    P = Pigment                                                                    O = Other characteristics                                                

                                      Table 4                                      __________________________________________________________________________     Growth on Carbon Compounds in the Synthetic Medium of                          Pridham and Gottlieb*                                                                       S. libani subsp. soldani                                                                            S. libani                                    __________________________________________________________________________                  NRRL 8173                                                                             NRRL 8174                                                                             NRRL 8175                                                                             UC-5629                                      CONTROL      +      (-)    (-)    (+)                                          __________________________________________________________________________     1.  D-Xylose +      +      +      +                                            2.  L-Arabinose                                                                             +      (+)    (+)    (+)                                          3.  Rhamnose +      (-)    (-)    (+)                                          4.  D-Fructose                                                                              +      +      +      +                                            5.  D-Galactose                                                                             +      +      +      +                                            6.  D-Glucose                                                                               +      +      +      +                                            7.  D-Mannose                                                                               +      +      +      +                                            8.  Maltose  +      +      +      +                                            9.  Sucrose  +      +      +      +                                            10. Lactose  +      (-)    (-)    +                                            11. Cellobiose                                                                              +      (-)    (-)    (+)                                          12. Raffinose                                                                               +      +      +      +                                            13. Dextrin  +      +      +      +                                            14. Inulin   +      (-)    (-)    (+)                                          15. Soluble Starch                                                                          +      +      +      +                                            16. Glycerol +      +      +      +                                            17. Dulcitol (+)    (-)    (-)    (+)                                          18. D-Mannitol                                                                              +      +      +      +                                            19. D-Sorbitol                                                                              (+)    +      +      +                                            20. Inositol +      +      +      +                                            21. Salicin  (-)    -      -      -                                            22. Phenol   -      -      -      -                                            23. Cresol   -      -      -      -                                            24. Na Formate                                                                              -      -      -      -                                            25. Na Oxalate                                                                              (+)    (-)    (-)    (-)                                          26. Na Tartrate                                                                             (+)    (-)    (-)    (-)                                          27. Na Salicylate                                                                           -      -      -      -                                            28. Na Acetate                                                                              (+)    (+)    (+)    (+)                                          29. Na Citrate                                                                              +      (+)    (+)    (+)                                          30. Na Succinate                                                                            +      (+)    (+)    (+)                                          __________________________________________________________________________      *Pridham, T. G. and D. Gottlieb. 1948. The utilization of carbon compound      by some Actinomycetales as an aid for species determination. J. Bacteriol      56: 107-114.                                                                   + = Good growth                                                                (+) = Moderate growth                                                          (-) = Poor growth                                                              - = No growth                                                            

                                      Table 5                                      __________________________________________________________________________     Growth on Carbon Compounds in the Synthetic Medium of                          Shirling and Gottlieb *                                                        __________________________________________________________________________              S. libani subsp. soldani                                                                            S. libani                                        CONTROL  NRRL 8173                                                                             NRRL 8174                                                                             NRRL 8175                                                                             UC-5629                                          __________________________________________________________________________     Negative +      -      -      +                                                (synthetic                                                                     medium only)                                                                   Positive +      +      +      +                                                (synthetic                                                                     medium with                                                                    D-glucose)                                                                     1.  Arabinose                                                                           +      +      +      ++                                               2.  Sucrose                                                                             ++     ++     ++     ++                                               3.  D-Xylose                                                                            ++     +      ++     +                                                4.  Inositol                                                                            ++     ++     ++     ++                                               5.  D-Mannitol                                                                          +      ++     ++     ++                                               6.  D-Fructose                                                                          +      +      ++     ++                                               7.  Rhamnose                                                                            +      ±   -      -                                                8.  Raffinose                                                                           ++     ±   ±   +                                                9.  Cellulose                                                                           -      -      -      -                                                __________________________________________________________________________      * Shirling, E. B. and D. Gottlieb. 1966. Methods for characterization of       Streptomyces species. Int. J. Syst. Bacteriol.                           

4-Thiouracil is produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood, also, that for the preparation of limited amounts surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer's yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers' solubles solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium.

Production of 4-thiouracil can be effected at any temperature conducive to satisfactory growth of the micro-organism, for example, between about 18° and 40° C., and preferably between about 20° and 28° C. Ordinarily, optimum production of the compound is obtained in about 3 to 15 days. The medium normally remains acidic during the fermentation. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of 4-thiouracil and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil, agar plug, stored frozen above liquid N₂, or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of 4-thiouracil, so long as a good growth of the microorganism has been obtained.

A variety of procedures can be employed in the isolation and purification of 4-thiouracil from fermentation beers, for example, solvent extraction, partition chromatography, silica gel chromatography, liquid-liquid distribution in a Craig apparatus, adsorption on resins, and crystallization from solvents.

In a preferred recovery process 4-thiouracil is recovered from its culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation. The antibiotic is then recovered from the filtered or centrifuged broth by extraction. For the extraction of 4-thiouracil from the filtered broth, water-immiscible organic solvents in which it is soluble, for example, chloroform, ethylene dichloride, ethyl acetate (preferred), and methylene chloride can be used. Advantageously, the extraction is carried on after the filtered beer is adjusted to an approximate pH 7.0 with a mineral acid. The solvent extracts are combined and evaporated to dryness under vacuum. Alternately, 4-thiouracil can be recovered from the filtered fermentation beer by use of a resin which comprises a non-ionic macroporous copolymer of styrene crosslinked with divinylbenzene. Resins of this type are disclosed in U.S. Pat. No. 3,515,717. Examples of this type of resin are Amberlite XAD-2, XAD-4, XAD-12. These resins can be obtained from the Rohm and Haas Chemical Company. Further, anion exchange resins can be used to recover 4-thiouracil from the filtered fermentation broth. Examples of such resins are Dowex-1 and Dowex-2 in the hydroxide form. These resins can be obtained from the Dow Chemical Company. When an anion exchange resin is used in the recovery process, the 4-thiouracil can be eluted from the resin by adding a polar, water-miscible solvent to the aqueous stream. Examples of such solvents are acetone, methanol, i-propanol, ethanol, dioxane or tetrahydrofuran. The organic solvent can then be removed in vacuo and the 4-thiouracil extracted from the aqueous using one or more of the above solvents. If a solid forms on removal of the organic solvent, then the product can be recovered by filtration or lyophilization. A further alternate recovery process is to pass the filtered beer over activated charcoal and eluting the 4-thiouracil, as described above.

Purification of the 4-thiouracil preparation, obtained as described above, can be achieved by the use of silica gel or alumina chromatography using a pair of compatible lipophilic and hydrophilic solvents such as CHCl₃ /MeOH, CHCl₃ /EtOH, EtOAc/acetone, BuOAc/MeOH, EtOAc/MeOH (preferred), and the like.

Hereinafter are described non-limiting examples of the process of the subject invention. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1

Part A. Fermentation

One agar plug of Streptomyces libani subsp. soldani, NRRL 8173, stored above liquid nitrogen, is used to inoculate each of a series of 500-ml Erlenmyer flasks each containing 100 ml of sterile seed medium consisting of the following ingredients:

    ______________________________________                                         Glucose monohydrate                                                                             25         g/liter                                            Pharmamedia *    25         g/liter                                            Tap water q.s.    1         liter                                              ______________________________________                                          * Pharmamedia is an industrial grade of cottonseed flour produced by           Traders Oil Mill Company, Fort Worth, Texas.                             

The seed medium presterilization pH is 7.2. The seed inoculum is grown for 3 days at 28° C. on a Gump rotary shaker operating at 250 r.p.m. and having a 21/2 inch stroke.

Seed inoculum (5%), prepared as described above, is used to inoculate a series of 500 ml fermentation flasks, each containing 100 ml of sterile fermentation medium consisting of the following ingredients:

    ______________________________________                                         Glycerol           10 g/liter                                                  Yeast Extract      2.5 g/liter                                                 Brer Rabbit Molasses                                                                              10 g/liter                                                  Tryptone *         5 g/liter                                                   Tap water q.s.     1  liter                                                    ______________________________________                                          * Difco Laboratories, Detroit, Michigan.                                 

The inoculated fermentation flasks are incubated at a temperature of 28° C. for 5 days while being shaken on a Gump rotary shaker operating at 250 r.p.m. and having a 21/2 inch stroke. Foaming in the fermentation flasks is controlled by the antifoam agent UCON (a synthetic defoamer supplied by Union Carbide, N.Y., N.Y.). A representative 5-day fermentation has the following titers of antibiotic in the fermentation broth:

    ______________________________________                                         Day            Assay, BU/ml                                                    ______________________________________                                         2              6.7                                                             3              15.0                                                            4              10.0                                                            5              16.0                                                            ______________________________________                                    

The assay is a disc plate biounit assay using the bacterium Bacillus subtilis grown on a synthetic medium. The synthetic medium has the following composition:

    ______________________________________                                         Na.sub.2 HPO.sub.4 .sup.. 7H.sub.2 O                                                                1.7       g                                               KH.sub.2 PO.sub.4    2.0       g                                               (NH.sub.4).sub.2 SO.sub.4                                                                           1.0       g                                               MgSO.sub.4           0.1       g                                               Glucose              2.0       g                                               Bacto Agar *         15.0      g                                               Distilled water      1         liter                                           Metallic ion stock solution **                                                                      1         ml                                              ______________________________________                                          * Bacto Agar obtained from Difco Laboratories, Detroit, Michigan.              ** Metallic ion stock solution consists of the following:                

    NaMoO.sub.4 .sup.. 2H.sub.2 O                                                                       200       μg/ml                                        CoCl.sub.2           100       μg/ml                                        CuSO.sub.4           100       μg/ml                                        MnSO.sub.4           2         mg/ml                                           CaCl.sub.2           25        mg/ml                                           FeCl.sub.2 .sup.. 4H.sub.2 O                                                                        5         mg/ml                                           ZnCl.sub.2 *         5         mg/ml                                           ______________________________________                                          * ZnCl.sub.2 has to be dissolved separately using a drop of 0.1N HCl for       10 ml of water. The stock solution is heated to bring all the compounds i      solution, kept standing for 24 hours, and sterile filtered.              

This medium is inoculated with a spore suspension of B. subtilis (1.5 × 10¹⁰ cells/ml) at a rate of 0.5 ml/liter. The beer samples are applied to 12.5 mm diameter adsorbent paper discs (0.08 ml/disc), which are then applied to the surface of the assay plate. The assay system is incubated overnight at 37° C., and the zones of inhibition are measured. The potency of the sample is related to the diameter of the inhibition zone by means of the usual standard curve.

A biounit (BU) is defined as the concentration of the antibiotic which gives a 20 mm zone of inhibition under the above assay conditions. Thus, if for example a fermentation beer, or other solution containing the antibiotic, needs to be diluted 1/100 to give a 20 mm zone of inhibition, the potency of such beer or solution is 100 BU per ml.

Part B. Recovery

Whole fermentation beer (800 ml) at pH 8.0 is filtered with the aid of diatomaceous earth as a filter aid after the adjustment to pH 7.0 with 3N HCl. The resulting 750 ml of filtrate is extracted twice with 2-750 ml portions of ethyl acetate. The organic phases are combined, washed with a saturated sodium chloride solution, dried with magnesium sulfate, filtered and concentrated on a rotary evaporator; yield 166.0 mg of a preparation of 4-thiouracil.

Part C. Purification

A preparation of 4-thiouracil (116 mg), prepared as described above, is dissolved in about 5 ml of ethyl acetate and the solution is injected via a syringe onto a size C Merck pre-packed column of silica gel. The column is developed isocratically at 5 ml/min. to tube -35 whereupon the solution is switched to 10:1 EtOAc:MeOH (v/v). The 25 ml fractions are assayed by dipping 12.7 mm pads and placing them onto agar trays seeded with Salmonella schottmuelleri. The trays are incubated for 18 hours at 32° C. and the zones are read. Tubes 23-28 which give zones ranging from 24-38 mm are combined. Concentration of this pool gives 112.2 mg. of essentially pure 4-thiouracil.

EXAMPLE 2

By substituting Streptomyces libani subsp. soldani, strain W, NRRL 8174for S. libani subsp. soldani, NRRL 8173 in Example 1, there is obtained 4-thiouracil which can be recovered in the essentially pure form by the procedures disclosed in Example 1.

EXAMPLE 3

By substituting Streptomyces libani subsp. soldani, strain P, NRRL 8175 for S. libani subsp. soldani, NRRL 8173 in Example 1, there is obtained 4-thiouracil which can be recovered in the essentially pure form by the procedures disclosed in Example 1. 

We claim:
 1. A process for preparing 4-thiouracil which comprises cultivating Streptomyces libani subsp. soldani, having the identifying characteristics of NRRL 8173 in an aqueous nutrient medium under aerobic conditions until substantial antibiotic activity is imparted to said medium.
 2. A process, according to claim 1, wherein said aqueous nutrient medium contains a source of assimilable carbohydrate and assimilable nitrogen.
 3. A process for recovering 4-thiouracil from a 4-thiouracil fermentation beer which comprises:1. filtering a 4-thiouracil containing fermentation beer to obtain a clear beer;
 2. extracting said clear beer with an organic solvent in which 4-thiouracil is soluble to obtain solvent extracts containing 4-thiouracil;
 3. chromatographing said solvent extract on a silica gel column to obtain fractions containing 4-thiouracil; and
 4. concentrating said fractions to give an essentially pure preparation of 4-thiouracil.
 4. A process, according to claim 3, wherein the silica gel column is eluted with a solvent system consisting of ethyl acetate and methanol (10:1).
 5. A process for preparing 4-thiouracil which comprises cultivating Streptomyces libani subsp. soldani, strain W, having the identifying characteristics of NRRL 8174 in an aqueous nutrient medium under aerobic conditions until substantial antibiotic activity is imparted to said medium.
 6. A process, according to claim 5, wherein said aqueous nutrient medium contains a source of assimilable carbohydrate and assimilable nitrogen.
 7. A process for preparing 4-thiouracil which comprises cultivating Streptomyces libani subsp. soldani, strain P, having the identifying characteristics of NRRL 8175 in an aqueous nutrient medium under aerobic conditions until substantial antibiotic activity is imparted to said medium.
 8. A process, according to claim 7, wherein said aqueous nutrient medium contains a source of assimilable carbohydrate and assimilable nitrogen. 